The 86-kilodalton IE-2 protein of human cytomegalovirus is a sequence-specific DNA-binding protein that interacts directly with the negative autoregulatory response element located near the cap site of the IE-1/2 enhancer-promoter

Abstract

The 86-kDa IE-2 protein of human cytomegalovirus is able to autoregulate its own expression via a short nucleotide sequence, termed the cis repression signal (CRS), that is located between the TATA box and the cap site of the IE-1/2 enhancer-promoter. Here we report that the 86-kDa IE-2 protein can interact directly with the CRS, thus demonstrating that IE-2 is a DNA-binding protein. This could be shown by both DNase I protection and gel retardation experiments using a procaryotically expressed IE-2 protein that was purified to near homogeneity. The interaction was sequence specific since a mutated form of the CRS that had previously been reported to be defective in mediating negative regulation could no longer compete for binding in DNase I protection experiments. In addition, an IE-2-reactive monoclonal antibody was able to elicit a supershift in gel retardation experiments, thus proving the presence of IE-2 within the protein-DNA complex. These results suggest that formation of a specific complex between an IE protein and a target sequence located near the cap site of its own gene promoter may be a common mechanism used by both alphaherpesviruses and betaherpesviruses to autoregulate IE gene transcription, although the sequence requirements differ between the two herpesviral subgroups.